Crossing America: Phylogenetics of Two Plethodontid Salamander Genera Across Their Highly Fragmented Ranges

Introduction and Goals

Plethodontidae is a diverse group of salamanders with most of their diversity located in North America, specifically east of the Great Plains in the United States, along the West Coast of the United States, and in the mountainous ranges of central and southern Mexico (AmphibiaWeb 2020). Members of Plethodontidae are lungless, and depend on moist conditions to survive; this combined with their small size (most species reach under 15 cm in length) greatly constrains their ability to disperse over large distances. Two genera, Aneides and Plethodon, have representatives both on the West Coast and east of the Great Plains, regions which are seperated by wide expanses of dry, highly unsuitable habitat for Plethodontids. Even more surprisingly, a single species in each genus is also present in isolated mountain ranges in New Mexico (Aneides hardii and Plethodon neomexicanus); similar, seemingly-suitable habitat is present in other nearby mountain ranges in New Mexico, and much more extensively in the Colorado Rockies to the north and the Sierra Madre Occidental to the southwest, yet neither genus has been documented in these areas in spite of extensive collecting by herpetologists (Petranka 2010). Plethodon and Aneides are each believed to be monophyletic, and they are not believed to be sister taxa (Vieites et al. 2011), yet no other genus in Plethodontidae is present in more than one of the three hotspots described above (eastern US, West Coast, southern/central Mexico), and no Plethodontids at all are found west of central Texas and the Great Plains, and east of the Great Basin and the Idaho Rockies, besides the aforementioned two species (Petranka 2010). The goal of my project is to better understand the evolutionary history of these two oddly-distributed genera. Specifically, I would like to verify that Aneides and Plethodon are truly each monophyletic as they are currently understood to be, and if they are, I would like to understand how each genus expanded across North America, by examining the phylogenetic relationships between the eastern, western, and New Mexican representatives of each genus. This would provide insight as to whether the New Mexican species are each relics of a more widespread distribution of Plethodontids across North America, or whether they represent radiations from either coast.

Methods

I obtained publicly-available DNA sequences for two genes, ND-4 (mitochondrial) and Rag-1 (nuclear) from the NCBI database for Aneides hardii, Plethodon neomexicanus, several representatives of each of Aneides and Plethodon from both their eastern and western distributions, a number of outgroups within Plethodontidae including species which range relatively far north in Mexico (Aquiloeurycea galeanae, Isthmura bellii), and one outgroup from outside of Plethodontidae (Amphiuma tridactylum). I created a maximum-likelihood gene tree for each of these genes using IQ-TREE, with a bootstrap sample of 1000 to generate support values. I also generated a third tree by inputting the direct concatenation of these two genes into IQ-TREE with the specifications described above.

Results

Below is the gene tree generated for the ND-4 gene. This tree suggests that western members of Aneides form a monophyletic group, with Aneides hardii sister to this group, and the eastern Aneides aeneus sister to the rest of the genus. It suggests that all western members of Plethodon form a monophyletic group, with Plethodon neomexicanus nested within this group, while all eastern members of Plethodon sampled form a monophyletic group sister to much of Plethodontidae.

Support values for edges close to the tips were generally high, with relationships within smaller clades reflecting those presented by previously published phylogenies (e.g. eastern Plethodon in Fisher-Reid 2011). However, support values for edges between larger clades (e.g. Plethodontidae excluding western Plethodon and Ensatina) were very poor. In particular, the polyphyly of Plethodon, the monophyly of western Plethodon, and the placement of Plethodon neomexicanus within the clade of western Plethodon are poorly supported, and the monophyly of Aneides excluding the eastern species Aneides aeneus is also not well-supported. Relationships between larger clades (e.g. genera) in this gene tree greatly contradict established relationships between Plethodontid genera (Vieites et al. 2011).

Below is the gene tree generated for the Rag-1 gene. This tree suggests that western members of Aneides form a monophyletic group, with Aneides hardii sister to this group, and the eastern Aneides aeneus sister to the rest of the genus. It suggests that all western members of Plethodon form a monophyletic group, with Plethodon neomexicanus nested within this group, and eastern members of Plethodon form a monophyletic group sister to the aformentioned clade of western and New Mexican Plethodon. This tree also suggests that Plethodon and Aneides are each monophyletic as entire genera.

Unlike the ND-4 tree, support values in the Rag-1 tree are high for most edges close to the root of the tree, and relationships between genera are more consistent with established relationships between Plethodontid genera (Vieites et al. 2011). The monophyly of western Aneides, the placement of Aneides hardii as sister to western Aneides, and the placement of the eastern Aneides aeneus as sister the the rest of Aneides (and hence the monophyly of Aneides) are all well-supported. The monophyly of Plethodon and the monophyly of the group of eastern Plethodon are highly supported, and the monophyly of the group of western and New Mexican Plethodon is somewhat well-supported, but the placement of Plethodon neomexicanus as nested within the western Plethodon is not well-supported.

Finally, below is the gene tree generated for the concatenation of the two genes.

In this tree, the monophyly of Aneides and Plethodon are each well-supported, and predicted relationships within these genera are very similar to those in the Rag-1 tree, with similar support values. As in the Rag-1 tree, relationships between larger clades were mostly consistent with previously published phylogenies (Vieites et al. 2011).

Discussion

The Rag-1 tree and combined tree demonstrate with high support the monophyly of each of Plethodon and Aneides. This suggest that the two present-day Plethodontids in New Mexico are indeed the result of multiple seperate Plethodontid radiations into the region. The monophyly of these two genera also indicates a shared ancestry between the New Mexican Plethodontids and either eastern or western clades, not Mexican clades of Plethodontids such as Isthamura, Aquiloeurycea, and Pseudoeurycea.

Strongly-supported relationships in the Rag-1 and concatenated trees (and moderate-to-weakly supported relationships in the ND-4 tree) suggest that Aneides hardii is sister to the group of western Aneides, with eastern Aneides sister to this broader group. This could either mean that Aneides hardii is the result of a radiation of Aneides from the west coast before the diversification of that group, or it could mean Aneides hardii diverged from the ancestor of present-day western Aneides when the genus had a more widespread distribution across the American west, perhaps soon after the ancestor of the eastern Aneides aeneus diverged from the ancestor of the rest of the genus’s extant members.

Strongly-supported relationships in all three trees indicate that the group of eastern Plethodon is monophyletic, and does not include Plethodon neomexicanus, ruling out the possibiliy of Plethodon neomexicanus being the result of a recent radiation from the east. Weakly-supported relations ships that are consistent in all three trees suggest that Plethodon neomexicanus is nested within the clade of western Plethodon; if we accept this relationship, then Plethodon neomexicanus is likely the result of a radiation of Plethodon from the west that occurred after some of the diversification of extant Plethodon in the Pacific Northwest. However, stronger support would be required to make this conclusion.

I am not sure whether to trust the concatenated gene tree, because I worry that the two genes (especially since one is nuclear and one is mitochondrial) may have significantly different rates or patterns of evolution which are not properly accounted for by the simplicity of the model chosen by IQ-TREE (TIM2 transition model with empirical base frequencies and free rate model of evolutionary rate heterogeneity with 4 categories). Going forward, I intend to investigate ways of partitioning the sequences to account for this.

The ideal, thorough way to address this research question would be to construct a Baysian time-calibrated tree with fossil data. Knowing the absolute times of divergence of the New Mexican, eastern, and western clades of Plethodon and Aneides would allow us to better understand the climatic conditions under which these radiations were able to occur accross large areas which are now inhospitable to Plethodontid salamanders. Past research indicates that the two genera diverged from related genera during the Eocene after the closure of the western interior seaway (Vieites et al. 2008). Since then, as the ancestors of extant species of Plethodon and Aneides diverged, the climate of the western United States has flutuated between periods of higher and lower rainfall, providing opportunites for Plethodontids to disperse across areas of the American west that now serve as barriers to them.

References

AmphibiaWeb. 2020. https://amphibiaweb.org University of California, Berkeley, CA, USA. Accessed 30 Nov 2020.

B.Q. Minh, H.A. Schmidt, O. Chernomor, D. Schrempf, M.D. Woodhams, A. von Haeseler, R. Lanfear (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol., 37:1530-1534. https://doi.org/10.1093/molbev/msaa015

Fisher-Reid, M. Caitlin. (2011). What are the consequences of combining nuclear and mitochondrial data for phylogenetic analysis? Lessons from Plethodon salamanders and 13 other vertebrate clades. BMC evolutionary biology. 11. 300. 10.1186/1471-2148-11-300. https://www.researchgate.net/figure/Combined-data-Plethodon-phylogeny-Phylogeny-of-the-salamander-genus-Plethodon-based-on-a_fig1_51717349

NCBI Resource Coordinators. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2018 Jan 4;46(D1):D8-D13. doi: 10.1093/nar/gkx1095. PMID: 29140470; PMCID: PMC5753372.

Petranka, James W. (2010). Salamanders of the United States and Canada. Smithsonian Books. ISBN: 9781588343086

Vieites, David & Min, Mi-Sook & Wake, David. (2008). Rapid diversification and dispersal during periods of global warming by plethodontid salamanders. Proceedings of the National Academy of Sciences of the United States of America. 104. 19903-7. 10.1073/pnas.0705056104. https://www.researchgate.net/publication/5771995_Rapid_diversification_and_dispersal_during_periods_of_global_warming_by_plethodontid_salamanders

Vieites, David & Nieto-Román, Sandra & Wake, Marvalee & Wake, David. (2011). A multigenic perspective on phylogenetic relationships in the largest family of salamanders, the Plethodontidae. Molecular phylogenetics and evolution. 59. 623-35. 10.1016/j.ympev.2011.03.012. http://ibdev.mcb.berkeley.edu/labs/wake/373_multigenic_Plethodontidae.pdf

Final Project

Phylogenetic Biology - Final Project